Methods for Modulating Macrophage Proliferation Using Polyamine Analogs

ABSTRACT

Methods for modulating macrophage proliferation in an individual afflicted with or at risk for a macrophage-associated disease are provided. The methods employ a polyamine analog, or salt or protected derivative thereof. Macrophage proliferation has been implicated in a number of serious disorders, including AIDS (HIV)-associated dementia, AIDS-associated non-Hodgkin&#39;s lymphoma, and Alzheimer&#39;s disease. The invention also provides methods for aiding diagnosis and monitoring therapy of a macrophage-associated non-HIV associated dementia, especially Alzheimer&#39;s disease. The invention also provides methods of delaying development of macrophage-associated non-HIV associated dementias, including Alzheimer&#39;s disease, which entail administration of an agent which modulates macrophage proliferation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.Nos. 60/063,317 and 60/063,318, both filed Oct. 27, 1997, and both ofwhich are incorporated by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made in part during work supported by a grant fromone or more government agencies. The government has certain rights inthe invention.

TECHNICAL FIELD

This invention relates to macrophage proliferation. More specifically,it relates to the use of polyamine analogs or salts or protectedderivatives thereof to modulate macrophage proliferation, particularlyin individuals afflicted with or at risk for a macrophage-associateddisease. The invention also relates to methods of aiding diagnosis,monitoring therapy, and delaying development of macrophage proliferationdisorders (in the context of non-HIV associated dementias, such asAlzheimer's disease) that entail detection and/or modulation ofmacrophage proliferation.

BACKGROUND ART

Cell proliferative diseases represent a major health problem and threatworldwide. Such diseases are characterized by the single or multipleabnormal proliferation of cells, groups of cells or tissue(s). See U.S.Pat. No. 5,665,588. Cell proliferative diseases include, but are notlimited to, AIDS dementia, various cancers, atherosclerosis,vitreoretinopathy, psoriasis, neurodegenerative disorders andnephropathy. See U.S. Pat. Nos. 5,639,600, 4,847,257 and 5,672,746.Dementias, or progressive mental failures, particularly present aserious worldwide health problem, both in terms of the extent ofdebilitation and health care costs. Dementias include non-HIV-associateddementias (such as Alzheimer's disease or AD) and HIV-associateddementia.

Macrophages are terminally differentiated cells generally incapable offurther cell division. Surprisingly, macrophage proliferation has beenimplicated in certain serious proliferative diseases such as lymphoma,cardiovascular disease, and nephrosclerosis. U.S. Pat. No. 5,639,600.Gabrielian et al. reported the role of macrophage infiltration intraumatic proliferative vitreoretinopathy. (1994) Curr. Eye. Res.13:1-9. McGrath et al. disclosed the involvement of clonally expandedmacrophages in the induction of cancerous tumor growth and AIDSdementia. U.S. Pat. Nos. 5,639,600 and 5,580,715; see also Pulliam etal. (1997) Lancet 349:692-695; McGrath et al. (1995) J. Acquired Imm.Def Syn. Hum. Retro. 8: 379-385; Shiramizu et al. (1994) Cancer Res.54:2069-2072.

AD is a degenerative brain disorder clinically characterized byprogressive loss of memory, cognition, reasoning, judgment and emotionalstability that gradually leads to profound mental deterioration andultimately death. AD is a common cause of dementia in the elderly and isbelieved to represent the fourth most common medical cause of death inthe United States. The disease is currently estimated to affect overfour million Americans. To date, the disease is incurable. Variousfactors such as genetic predisposition, infectious agents, toxins,metals, and head trauma have all been suggested as possible mechanismsof AD neuropathology. A prevailing theory explaining the cause of AD isrelated to abnormal deposition of

-amyloid into plaques with associated neurofibrillary tangles. No HIV orother infectious agent has to date been associated with AD, and ADpathologic lesions are different than those associated with AIDSdementia.

Several different classes of chemical compounds have been reported thatcan inhibit abnormal cellular proliferation. These classes include, forexample, anionic oligomers, amino 1,2,3-triazoles, valproic acidderivatives and polyamines. Cardin et al. reported that certain anionicoligomers possess antiproliferative activity. U.S. Pat. No. 5,460,807.Water soluble polyureas and polyamides with a molecular weight of lessthan 10,000 inhibit smooth muscle cell proliferation in culture and invivo. Cardin et al. state that the anionic oligomers can be used in thetreatment of atherosclerosis. Hupe et al. disclosed that certaintriazoles are antiproliferatives. U.S. Pat. No. 4,847,257. Amino 1, 2, 3triazoles inhibit labeled thymidine incorporation into intact pig skinand also inhibit keratinocyte proliferation. Hupe et al. state that thetriazoles can be used in the treatment of psoriasis, a chronic skindisease which is characterized by hyperproliferation of the epidermis.Nau et al. discovered that certain acids inhibit cell mitosis. U.S. Pat.No. 5,672,746. Derivatives of valproic acid decrease neuro-2a cellproliferation in vitro. Nau et al. state that the valproic acidderivatives can be used for the prevention and treatment ofneurodegenerative disorders such as Alzheimer's disease. The treatmentwould be aimed at preventing the adverse effects of the disease bydirectly inhibiting pathologic neural cell growth.

The level of polyamines is intimately related to cell proliferation.Cellular levels of polyamines are carefully regulated by opposingsynthetic and catabolic pathways. Compounds that are able to lowerpolyamine levels are proposed for use in the treatment of rapidlyproliferating host cells such as cancer and psoriasis. A key polyaminecatabolizing enzyme spermidine-spermine N1-acetyltransferase (SSAT) isamong the few genes known to be inducible by the natural polyamines.Certain polyamine analogs exaggerate this response.1,11-diethylnorspermine (DENSPM) increases SSAT mRNA levels in humanmelanoma cells up to 20-fold, with an increase in immunodetectable SSATprotein by 300-fold. By comparison, natural polyamine spermine is farless effective, increasing SSAT mRNA by ˜3-fold and immunodetectableprotein by ˜7-fold. Fogel-Petrovic et al. (1996) Biochemistry 35:14435.Polyamine analogs also induce Z-DNA structure in vitro. This propertycorrelates inversely with the effects on cis-diaminedichloroplatinum(II) (CDDP) cytotoxicity in human brain tumor cells. Basu et al. (1996)Anticancer Res. 16:39.

U.S. Pat. No. 5,498,522 outlines the use of SSAT as a prognosticindicator or tumor response marker. Either SSAT enzyme activity, SSATenzyme protein, or mRNA transcripts can be measured directly, or otherdeterminants related to SSAT induction can be measured, such as SSATco-factor acetylCoA, and the SSAT products N1-acetylspermine andN1-acetylspermidine. Measurement of these determinants is proposed as aprognostic indicia and tumor response marker to evaluate the clinicaleffectiveness of anticancer agents comprising polyamine analogs.Determination is performed by collecting tumor cells by biopsy before orafter treatment, and measuring the selected determinant. Hibasami et al.[(1989) Cancer Res. 49:2065] prepared an inhibitor of the naturalpolyamine synthetic pathway. The compound,methylglyoxal-bis(cyclopentylamidinohydrazone) (MGBCP) inhibitedS-adenosylmethionine decarboxylase, spermine synthase, and sperminesynthetase, competing with S-adenosylmethionine, spermidine, andputrescine, respectively. MGBCP depleted spermidine and spermine inleukemic ascites cells, and prolonged survival time of mice bearing P388leukemia.

U.S. Pat. No. 5,541,230 (Basu et al.) indicates that sperminederivatives decrease growth in a number of human tumor cell lines, andpropose their use in cancer chemotherapy. Bergeron et al. (CancerChemother. Pharmacol.) showed that the polyamine analogs1,14-bis(ethylamino)-5,10-diazatetradecaone (BE-4-4-4), and1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) directlyaffects growth, survival, and cell cycle progression in human braintumor cell lines. The synthesis of BE-4-4-4-4 is disclosed in U.S. Pat.No. 5,541,230. U.S. Pat. No. 5,516,807 (Hupe et al.) claims a method fortreating vascular proliferative disorders following balloon angioplasty.Bis-ethyl norspermine was found to inhibit the growth of rat aorticsmooth muscle cells in culture for 8 to 15 days. It is proposed thatrestenosis following balloon angioplasty, graft, shunt, or athereotomycan be treated or prevented by administration of an effective amount ofa polyamine, although no such experiments were performed. For otherpublications relating to the synthesis and use of certain polyamines,the reader is referred to EP 277,635, EP 162,413, EP 399,519, JP85/6348, and U.S. Pat. No. 5,679,682; and to Bellevue et al. (1996)Bioorg. Med. Chem. Lett. 6:2765, and Porter et al. (1992) Falk Symposium62:201; Marton and Pegg (1995) Ann Rev. Pharmacol. Toxicol. 35:55-91.

What is needed are methods of modulating macrophage proliferation thatis associated with disease. There is also a need for methods ofindicating development and/or progression of non-HIV-mediated dementiasassociated with macrophage proliferation.

All publications cited herein are hereby incorporated in their entirety.

DISCLOSURE OF THE INVENTION

The present invention provides methods for modulating macrophageproliferation using a composition comprising a polyamine analog or saltor protected derivative thereof (or using a polyamine analog or salt orprotected derivative thereof), preferably in an individual afflictedwith or at risk for a disease with which macrophage proliferation isassociated, wherein the composition (or compound) is administered in anamount sufficient to modulate macrophage proliferation. In oneembodiment, the disease is a non-HIV-associated dementia, particularlyAlzheimer's disease. In another embodiment, the individual isHIV-infected and the disease is HIV-associated, such as HIV-associateddementia. Preferably, all the nitrogens of the polyamine analog aresecondary, tertiary, or quarternary amino groups. The composition canfurther comprise a pharmaceutically acceptable excipient. In someembodiments, the composition comprises mitoguazone dihydrochloride.

In another aspect, the invention provides methods for modulatingmacrophage proliferation in an individual comprising administering acomposition comprising an effective amount of an agent (or administeringan effective amount of an agent) which interferes with polyamineinteraction with a target in a proliferating macrophage, such as DNA,RNA, and/or membranes.

In another aspect, the invention provides methods of aiding diagnosis ofa macrophage-associated dementia, particularly a non-HIV-associateddementia, including Alzheimer's disease (AD) an individual, comprisingthe step of detecting the presence of and/or the level of proliferatingmacrophages in the individual.

In another aspect, the invention provides methods of monitoring therapyof a macrophage-associated dementia, particularly a non HIV associateddementia, in an individual comprising detecting the presence of, and/orthe level of, proliferating macrophages in a biological sample from theindividual. The invention also includes methods of monitoring anindividual at high risk of developing macrophage-associated, non-HIVassociated dementia (such as AD), comprising detection of the presenceof, or the level of, proliferating macrophages in a biological samplefrom that individual.

In another aspect, the invention provides methods of modulatingmacrophage proliferation in an individual afflicted with or at risk forAlzheimer's disease comprising administering to the individual acomposition comprising a compound selected from the group consisting ofa polyamine analog, a salt of a polyamine analog, and a protectedderivative of a polyamine analog (or administering an effective amountof a compound selected from the group consisting of a polyamine analog,a salt of a polyamine analog, and a protected derivative of a polyamineanalog), wherein all nitrogen atoms of said polyamine analog aresecondary, tertiary, or quartenary amino groups, and where thecomposition (or compound) is administered in an amount sufficient tomodulate macrophage proliferation in the individual.

In another aspect, the invention provides methods of modulatingmacrophage proliferation in an individual at afflicted with or at riskfor a macrophage-associated non-HIV dementia (such as in a non-HIVinfected individual) other than AD, comprising administering to theindividual an effective amount of an agent that modulates macrophageproliferation (or a composition comprising an effective amount of anagent that modulates macrophage proliferation), wherein an effectiveamount is an amount sufficient to modulate macrophage proliferation. Insome embodiments, the agent is a polyamine analog(s) as describedherein.

In another aspect, the invention provides methods of delayingdevelopment of a macrophage-associated dementia other than AD in anon-HIV-infected individual comprising administering to an individual aneffective amount of an agent that modulates macrophage proliferation (oradministering a composition comprising an effective amount of theagent). The agent may be, for example, a polyamine analog.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are graphs denoting percent inhibition or killing invitro of macrophage proliferation by varying concentrations of polyamineanalogs DEHOP (SunPharm) and BE-4444, compared to antineoplastic agentcyclosphosphamide. Blood was obtained from patients with AIDS dementia.

FIG. 2 is a bar graph denoting comparative in vitro cell toxicity ofmacrophage supernatants which have been treated (Rx) or not treated(uRx) with the polyamine analog DEHOP. Designations are as follows:Cont., control; D-uRx, AIDS dementia, no DEHOP; D-Rx, AIDS dementia,DEHOP; L-uRx, AIDS lymphoma, no DEHOP; L-Rx, lymphoma, DEHOP.

FIG. 3 is a graph depicting the effects of polyamine analogs DENOP andSL-11037 on macrophage proliferation, as evidenced by PCNA expression inCD14 cells. Macrophages were obtained from peripheral blood of patientswith AD.

FIG. 4 is a bar graph depicting detection of proliferating macrophages(PCNA/CD14 cells) in the peripheral blood of patients with AD ascompared to non-disease and disease controls. C: control (N=6); MS:multiple sclerosis (N=2); PAR: Parkinson's disease (N=2); ALS:Amyotrophic lateral sclerosis (N=2); ADC: AIDS dementia complex (N=7);ALZ: AD (N=6).

FIG. 5 is a graph depicting polyamine analog-mediated inhibition of PCNAmacrophage detection from peripheral HIV⁺ sarcoid patients, usingpolyamine analogs SL-11047, SL-11048, and SL-11044.

FIG. 6 is a graph depicting PCNA⁺/CD14 cell survival after treatmentwith polyamine analogs DEHOP and SL-11047. N indicates the number ofexperiments performed.

FIG. 7 is a graph showing the in vivo responses to DEHOP of CD14⁺ cellsin a peripheral blood mononuclear cell preparation (PBMC) from the 2AIDS lymphoma patients, FL and LT.

MODES FOR CARRYING OUT THE INVENTION

We have discovered that polyamine analogs are particularly effective inmodulating macrophage proliferation. We have also discovered elevated,abnormal levels of macrophage proliferation in peripheral blood ofpatients with Alzheimer's disease (AD). We have also observed thatmacrophage proliferation is associated with a number of other seriousdisorders, including AIDS dementia, AIDS-associated non-Hodgkin'slymphoma, cardiovascular disease and nephrosclerosis. U.S. Pat. No.5,639,600; see also commonly owned U.S. provisional application U.S.Ser. No. 60/063,318. Without wishing to be bound by a particular theory,we note that this macrophage proliferation phenomenon is associatedwith, and may contribute to the disease sequelae of, non-HIV associateddementias such as AD.

That polyamine analogs are effective in modulation of macrophageproliferation is evidenced by a polyamine analog-mediated decrease inproliferation marker PCNA (proliferating cell nuclear antigen) in CD14expressing cells (i.e., macrophages). We have also observed thatsupernatants of proliferating macrophages are toxic to cells in thecontext of AIDS dementia, particularly brain cells. Controlling unwantedand harmful macrophage proliferation is thus a crucial aspect ofdeveloping new, effective treatment modalities for these disorders.

Accordingly, the invention provides methods for modulating macrophageproliferation, which are useful for controlling, palliating, and/ordelaying development of these macrophage-proliferative disorders,including, but not limited to, certain neoplasms, HIV-associateddiseases, and non-HIV associated dementias such as AD. The inventionalso provides methods of modulating macrophage proliferation inindividuals afflicted with or at risk for a non-HIV associated dementia(as well as methods of delaying development of such dementia(s)), otherthan AD, which employ an agent which modulates macrophage proliferation(preferably a polyamine analog). The invention also provides methods ofaiding diagnosis and/or monitoring therapy which entail measuring thepresence of proliferating macrophages.

As discussed below, preferred agents for modulation of macrophages arepolyamine analogs especially 1,11-bis(ethyl)norspermine;1,8-bis(ethyl)spermidine (BES); 1,12-bis(ethyl)spermine (BESm; DESPM(N¹, N¹²-diethylspermine); 1,11-bis(ethylamino)-4,8-diazaundecane(BE-3-3-3); 1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4)(Diethylhomospermine, N¹, N¹⁴-diethylhomospermine; DEHOP or DEHSPM);diethyl-norspermine (DENOP);1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4);N-ethyl-N′-(2-(3′-ethylamino-propylaminomethyl)-cis-cyclopropylmethyl)-propane 1,3-diamine tetrahydrochloride(SL-11037); N-ethyl-N′-(2-(3′-ethylamino-propylaminomethyl)-trans-cyclobutylmethyl)-propane 1,3-diamine tetrahydrochloride(SL-11038); N-ethyl-N′-(2-(3′-ethylamino-propylaminomethyl)-trans-cyclopropylmethyl)-propane 1,3-diamine tetrahydrochloride(SL-11044; and N,N′-bis(3-ethylaminopropyl)-cis-but-2-ene-1,4-diaminetetrahydrochloride (SL-11047).

DEFINITIONS

As used herein, the terms “macrophage” and “monocyte” are usedinterchangeably, as it is understood that in the art the term “monocyte”is often used to describe a circulating mononuclear cell that expressesthe CD14 cell surface marker, and when in a tissue this cell is alsoclassified as a macrophage.

A “proliferating macrophage” is a term understood in the art and as usedherein denotes a macrophage which is dividing. Normally a macrophage isa terminally differentiated cell incapable of further division. Forpurposes of this invention, a “proliferating macrophage” is capable offurther division or is in a portion of the cell cycle not considered tobe terminal or end stage. Preferably, the proliferation is clonal, i.e.,is derived from a single cell. Methods of detecting proliferatingmacrophage(s) is discussed below.

As used herein, detecting the “presence of proliferating macrophages”generally means detecting the level of proliferating macrophages. It isunderstood that an absolute or even relative level need not bedetermined; an observation of detectable proliferating macrophages issufficient.

A “macrophage-associated” disease, disorder or indication is a disease,disorder or indication that is associated with an elevated, or abnormal,level of macrophage proliferation as compared to control sample(s). Suchdisorders include, but are not limited to, AIDS-associated dementia,Alzheimer's disease (AD), AIDS lymphoma, follicular lymphoma, mycosesfungoides, T cell and B cell lymphomas with significant macrophagecompartments, atherosclerosis, kidney disease such as focal segmentalglomerulosclerosis and membrane proliferative glomerulo nephropathy,psoriaform dermatitities, AIDS-associated diarrhea, prelymphomatisautoimmune disease such as AILD (angioimmunoblastic lymphadenophathywith dysproteinemia) and herpes virus associated diseases such asCastleman's disease and Kaposi's sarcoma. The terms “disorder” and“disease” are used interchangeably herein. “Macrophage-associateddementia” is a dementia that is associated with an elevated, orabnormal, level of macrophage proliferation as compared to controlsample(s). Such dementias include, but are not limited to, AD. Amacrophage-associated disorder, disease or dementia can be HIV-mediatedor non-HIV-mediated, or HIV-associated or non-HIV associated. A“non-HIV-mediated” disease or dementia is a disease or dementia which isnot caused by HIV, either directly or indirectly. A “non-HIV-associated”disease or dementia is a disease or dementia not normally associatedwith or secondary to HIV infection. An “HIV-mediated” disease, dementiaor indication is directly or indirectly caused by (and/or linked to) HIVinfection. An “HIV-associated” disease, dementia or indication isdefined more broadly as generally associated with or secondary to an HIVinfection; “HIV-mediated” diseases, for example, are included in thoseconsidered to be “HIV-associated.”

An individual “afflicted with” a macrophage associated disorder ornon-HIV associated dementia means that the individual has been diagnosedas having, or is suspected as having, a macrophage associated disorderor a non-HIV associated dementia.

By a “polyamine”, a term well-understood in the art, is meant any of agroup of aliphatic, straight-chain amines derived biosynthetically fromamino acids; polyamines are reviewed in Marton et al. (1995) Ann. Rev.Pharm. Toxicol. 35:55-91. By “polyamine” is generally meant anaturally-occurring polyamine or natural polyamine, which are naturallyproduced in eukaryotic cells. Examples of polyamines include putrescine,spermidine, spermine and cadaverine.

By “polyamine analog” is meant an organic cation structurally similarbut non-identical to naturally-occurring polyamines such as spermineand/or spermidine and their precursor, diamine putrescine. Polyamineanalogs can be branched or un-branched, or incorporate cyclic moieties.Examples of polyamine analogs include, without limitation,N¹,N¹⁴-diethylhomo-spermine (DEHSPM) and N¹,N¹²-diethylspermine (DESPM).See, for example, WO 98/17624 and U.S. Pat. No. 5,541,230. U.S. Pat.Nos. 5,037,846 and 5,242,947 disclose polyamines comprising primaryamino groups. Especially preferred are polyamine analogs wherein allnitrogen atoms of said polyamine analogs are independently secondary,tertiary, or quartenary amino groups.

An “alkyl” is a cyclic, branched, or straight chain chemical groupcontaining carbon and hydrogen, such as methyl, butyl, t-butyl, pentyl,cyclopropyl, and octyl. Alkyl groups can be either unsubstituted orsubstituted with one or more substituents, e.g., halogen, alkoxy,acyloxy, amino, hydroxyl, mercapto, carboxy, benzyl. Alkyl groups can besaturated or unsaturated (e.g., containing —C═C— or —C≡C— subunits), atone or several positions.

Unless otherwise specified, alkyl groups will comprise 1 to 8 carbonatoms, preferably 1 to 6, and more preferably 1 to 4 carbon atoms.“Cycloalkyl” refers to cyclic alkyl groups only, such as cyclopropyl,cyclobutyl, cyclopentyl, etc. “n-alkyl” refers to a linear (i.e.,straight-chain) alkyl group only, while “branched alkyl” refers tobranched alkyl groups to the exclusion of cyclic and linear alkylgroups. “Alkenyl” refers to a cyclic, branched, or straight chainchemical group containing carbon and hydrogen where at least one bond ismonounsaturated, such as ethenyl, cyclopentenyl, or 1,3-butadienyl.Alkenyl groups can be substituted as indicated for alkyl groups. Alkenylgroups can be designated as cyclic, linear (n-alkenyl) or branched in ananalogous fashion to the preceding designations for alkyl. An “aryl” isan unsaturated aromatic carbocyclic group having a single ring (e.g.,phenyl), or multiple condensed rings (e.g., naphthyl), which canoptionally be unsubstituted or substituted with amino, hydroxyl, alkyl,alkoxy, chloro, halo, mercapto and other substituents.

A “stereoisomer” is defined as any optical isomer of a compound,including enantiomers and diastereomers. Unless otherwise indicated,structural formula of compounds are intended to embrace all possiblestereoisomers.

A “salt” is defined as a compound formed by the replacement of one ormore hydrogen atoms with elements or groups, which is composed of anionsand cations, which usually ionizes in water; a salt is formed, forinstance, by neutralization of an acid by a base.

A polyamine analog salt can comprise, for example, chloride ions.

“Protected derivative” is used to refer to a compound protected with aprotecting group. “Protecting group” refers to a chemical group thatexhibits the following characteristics: 1) reacts selectively with thedesired functionality in good yield (preferably at least 80%, morepreferably at least 90%, more preferably at least 95%, still morepreferably at least 99%) to give a protected substrate that is stable tothe projected reactions for which protection is desired; 2) isselectively removable from the protected substrate to yield the desiredfunctionality; and 3) is removable in good yield (preferably at least80%, more preferably at least 90%, more preferably at least 95%, stillmore preferably at least 99%) by reagents compatible with the otherfunctional group(s) present or generated in such projected reactions.Examples of suitable protecting groups can be found in Greene et al.(1991) Protective Groups in Organic Synthesis, 2nd Ed. (John Wiley &Sons, Inc., New York). Exemplary protecting groups for the aminofunctionality include, but are not limited to, mesitylenesulfonyl(MesSO₂), benzyloxycarbonyl (CBz), t-butyloxycarbonyl (Boc),t-butyldimethylsilyl (TBDIMS), 9-fluorenylmethyloxycarbonyl (Fmoc), orsuitable photolabile protecting groups such as 6-nitroveratryloxycarbonyl (Nvoc).

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals, sportanimals, rodents, primates, and pets. A “non-HIV-infected individual” isan individual who has not been infected by HIV. An “HIV-infected”individual may or may not yet display clinical manifestations ofinfection. HIV and methods of detecting HIV infection are well known inthe art and need not be discussed herein.

As used herein, “biological sample” encompasses a variety of sampletypes obtained from an individual and can be used in a diagnostic ormonitoring assay. The definition encompasses blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom, and the progenythereof. The definition also includes samples that have been manipulatedin any way after their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. Generally,the sample will be, or be derived from, peripheral blood. Preferably,the blood will have been enriched for a macrophage fraction, by using,for example, glass or plastic adherence.

As used herein, “aiding diagnosis” means that these methods assist inmaking a clinical determination regarding the classification, or nature,of the dementia, and may or may not be conclusive with respect to thedefinitive diagnosis. The method of aiding diagnosis of amacrophage-associated disease can comprise the step of detecting thelevel of proliferating macrophages in a biological sample from theindividual, wherein the disease is dementia, such as anon-HIV-associated dementia, such as Alzheimer's disease. Dementias mayor may not be associated with clonal macrophage proliferation, andmaking this classification may assist in developing and recommendingtreatment strategies as well as evaluating prognosis.

“Development” of a dementia herein means initial manifestations and/orensuing progression of the disorder. Development of disease can bedetectable and assessed using standard clinical techniques, such asneurofunction/cognitive tests and brain scanning technologies such asMRI. However, development also refers to disease progression that may beundetectable. For purposes of this invention, progression refers to thebiological course of the disease state. “Development” includesoccurrence, recurrence, and onset. As used herein “onset” or“occurrence” of a neurological disorder includes initial onset and/orrecurrence. As used herein, “delaying” development of disease means todefer, hinder, slow, retard, stabilize, and/or postpone development ofthe disease. This delay can be of varying lengths of time, depending onthe history of the disorder and/or the medical profile of the individualbeing treated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop detectable disease. A method that “delays”development of disease is a method that reduces the extent of thedisease in a given time frame, when compared to not using the method.Such comparisons are typically based on clinical studies, using astatistically significant number of subjects, although this knowledgecan be based upon anecdotal evidence. “Delaying development” can meanthat the extent and/or undesirable clinical manifestations are lessenedand/or time course of the progression is slowed or lengthened, ascompared to not administering the agent. Thus the term also includes,but is not limited to, alleviation of symptoms, diminishment of extentof disease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, and remission (whether partial or total)whether detectable or undetectable.

As used herein, an “effective amount” (e.g., of an agent) is an amount(of the agent) that produces a desired and/or beneficial result. Aneffective amount can be administered in one or more administrations. Forpurposes of this invention, an effective amount is an amount sufficientto produce modulation of macrophage proliferation. An “amount (of apolyamine analog) sufficient to modulate macrophage proliferation”preferably is able to alter the rate of proliferation of macrophages byat least 25%, preferably at least 50%, more preferably at least 75%, andeven more preferably at least 90%.

Such modulation may have desirable concomitant effects, such as topalliate, ameliorate, stabilize, reverse, slow or delay progression ofdisease, delay or even prevent onset of disease.

As used herein, the term “agent” means a biological or chemical compoundsuch as a simple or complex organic or inorganic molecule, a peptide, aprotein or an oligonucleotide. A vast array of compounds can besynthesized, for example oligomers, such as oligopeptides andoligonucleotides, and synthetic organic compounds based on various corestructures, and these are also included in the term “agent”. Inaddition, various natural sources can provide compounds, such as plantor animal extracts, and the like. Agents include, but are not limitedto, polyamine analogs. Agents can be administered alone or in variouscombinations.

“Modulating” macrophage proliferation means that the rate ofproliferation is altered when compared to not administering an agentthat interferes with natural polyamine interaction with DNA (including,but not limited to, interfering with a polyamine biosynthetic pathway,interfering with the intracellular concentration of spermidine,competitors, inhibitors of DNA interaction by a natural polyamine,interfering with polyamine metabolism, etc.), such as a polyamineanalog. Preferably, “modulating” macrophage proliferation means a changein the rate of macrophage proliferation of at least 25%, preferably atleast 50%, more preferably at least 75%, and even more preferably atleast 90%. Generally, for purposes of this invention, “modulating”macrophage proliferation means that the rate of proliferation isdecreased when compared to the rate of proliferation in that individualwhen no agent is administered. However, during the course of therapy,for example, it may be desirable to increase the rate of proliferationfrom a previously measured level. The degree of modulation may beassessed by measurement of macrophage proliferation, which will bediscussed below, and generally entails detecting a proliferationmarker(s) in a macrophage population or uptake of certain substancessuch as BrdU or ³H-thymidine (which would provide a quantitative measureof proliferation). Further, it is possible that, if the macrophages areproliferating due to a genetic alteration (such as transposition,deletion, or insertion), this alteration could be detected usingstandard techniques in the art, such as RFLP (restriction fragmentlength polymorphism).

A “target” of a polyamine or polyamine analog is an entity whichinteracts, either directly or indirectly, with the polyamine orpolyamine analog(s). Examples of targets are DNA, RNA, and/or membranes.

Methods of the Invention

The invention provides methods for modulating macrophage proliferationin an individual afflicted with or at risk for a macrophage-associateddisease comprising administering a polyamine analog, a salt of apolyamine analog, or a protected derivative of a polyamine analog, in anamount sufficient to modulate macrophage proliferation in the individual(i.e., an effective amount). Alternatively, a composition comprising apolyamine analog, or a protected derivative of a polyamine analog isadministered in an amount sufficient to modulate macrophageproliferation (i.e., an effective amount). Examples ofmacrophage-associated diseases have been described above, and include,but are not limited to, HIV-associated diseases (i.e., in anHIV-infected individual) such as HIV associated dementia, non HIVassociated dementias (such as AD), and certain neoplasms. Polyamineanalogs are discussed below.

For purposes of this invention, an individual suitable foradministration of a polyamine analog (or, in certain contexts, such asnon-HIV associated dementia other than AD, an agent which modulatesmacrophage proliferation) is one who has been diagnosed as having amacrophage-associated disorder, such as AIDS dementia, AIDSnon-Hodgkin's lymphoma, Alzheimer's disease, or who is adjudged to be athigh risk for developing such a disorder. In some embodiments, anindividual suitable for administration of an agent which modulatesmacrophage proliferation is one who has been diagnosed as having amacrophage-associated, non HIV associated dementia other than AD, or whois adjudged to be at risk for developing such a dementia. As is evidentto one skilled in the art, these methods can apply to those individualsnot displaying any symptoms. An “at risk” or “high risk” individual isan individual who has a discrete and significant risk of developingdisease (a macrophage-associated disorder). An “at risk” or “high risk”individual may or may not have detectable disease, and may or may nothave displayed detectable disease prior to receiving the method(s)described herein. “High risk” (or “at risk”) denotes that an individualhas one or more so-called risk factors, which are measurable parametersthat correlate with development of disease. An individual having one ormore of these risk factors has a higher probability of developingdisease than an individual without these risk factor(s). These riskfactors include, but are not limited to, genetic (i.e., hereditary)considerations (including family history and genetic markers), andpresence or absence of appropriate chemical markers and exposure toenvironments, conditions, or factors which would increase thepossibility of acquiring a particular disease. Retroviral infections,especially retroviral insertions into particular genetic loci (such asfur or PDGF), may also be considered a risk factor. A high riskindividual has one, preferably two, more preferably three, risk factors.However, it is understood that having only one risk factor can oftenindicate high risk.

Because all risk factors for developing macrophage-associated diseaseare not known, and the interplay among these factors (in terms ofoverall risk) are not fully understood, it is clear to one skilled inthe art that individuals suitable for administration of an agent forpurposes of this invention can have clinical features in common, andthat individuals not falling clearly in the categories described abovecan nonetheless be considered suitable candidates for administration ofan agent. For example, an individual having a genetic marker fordevelopment of a neurodegenerative disorder (such as the apoE gene)could be considered at risk for developing Alzheimer's disease, eventhough no previous disease has been observed. In this context,administration of an agent to such an individual could result in delayof occurrence of disease, even to the extent that the individual doesnot develop AD within his or her lifetime (or develops it later thanwould have been expected). Another example is an individual who is beingtreated using other modes of therapy, and who is showing clinicalresponsiveness to the therapy (i.e., stabilization or remission). Suchan individual may be adjudged as at “high risk” even though the initialcourse of therapy is not yet completed, due to projection of clinicalprogress by the clinician, and can be a suitable candidate for receivingan agent before completion of the initial therapy. The clinician, as oneskilled in the art, has discretion to determine whether treatment usingan agent may be indicated.

In another embodiment, the invention provides methods for modulatingmacrophage proliferation in an individual (who is generally afflictedwith or at risk of for a macrophage associated disease) comprisingadministering a composition comprising an effective amount of an agentthat interferes with polyamine interaction with proliferating macrophagetarget, such as DNA, RNA, and/or membranes. An agent that interfereswith polyamine interaction with a proliferating macrophage target(s) isone which interferes with any aspect of natural polyamine synthesisand/or metabolism, intracellular concentration regulation, and/orfunction (i.e., interaction with DNA).

The present invention further provides methods for aiding in thediagnosis of or monitoring therapy in individuals having amacrophage-associated disease, wherein the disease is a non-HIVassociated dementia, such as AD. These methods comprise detecting thepresence of proliferating macrophages in a biological sample from theindividual.

In those individuals considered at high or significant risk ofdeveloping dementia, detection of proliferating macrophages in abiological sample may also assist in alerting the individual and/or theclinician of possible precursor disease. Thus, the invention alsoincludes methods of monitoring an individual at risk or high risk ofdeveloping dementia, comprising detection of proliferating macrophagesin a biological sample from that individual. Preferably, the individualis “afflicted with” (e.g., diagnosed as having, suffering from and/ordisplaying one or more clinical symptoms of) a particular disease,disorder or indication, or at “risk” for (e.g., having a geneticpredisposition for, or family history of, or being environmentallyexposed to factors which increase the probability of acquiring) aparticular disease, disorder, or indication.

In another embodiment, the invention provides methods of monitoringtherapy of a macrophage-associated dementia, which is a non-HIVassociated dementia, such as AD, in an individual (who is generally notinfected with HIV, although it is possible to develop a non-HIVassociated dementia in an individual who is also infected with HIV)comprising detecting the presence of (i.e., the level of) proliferatingmacrophages in a biological sample. As the level of macrophageproliferation is associated with these conditions, monitoring theselevels may in turn indicate initial responsiveness and efficacy, as wellas the appropriate dosage of the therapy. It is understood thatmonitoring therapy or an individual at (high) risk means that biologicalsample(s) are obtained at different times, for example, duringapplication of therapy, and are compared, either with each other, acontrol, and/or a desired value. In one embodiment, monitoring therapyincludes the step of detecting macrophage proliferation.

Detection of proliferating macrophage(s) can be achieved using any ofseveral techniques. In some embodiments of the invention, proliferationis measured in relation to circulating macrophages, and is performed ona leukocyte preparation from peripheral blood. In other embodiments ofthe invention, proliferation is measured in relation to tissue-fixedmacrophages, typically performed on tissue sections. Proliferatingmacrophages may be detected, for example, by assaying cell proliferativemarkers, such as PCNA, Ki67 or uptake of bromodeoxyuridine (BrdU) or³H-thymidine. These markers are distinct from those that identify only“activated” macrophages (as opposed to proliferating macrophages), suchas CD69 and CD25. The cellular subset representing macrophages may inturn be identified by detection of certain cell specific markers, suchas CD14, CD68, CD16, or nonspecific esterase. Detection of thesecell-type and/or proliferative markers use methods standard in the art,such as staining techniques and FACS sorting and analysis. These methodsare further described in Example 1. Further, it is possible that theseproliferating macrophages could be distinguished based on othercharacteristics, such as cell density (as measured in PERCOLL™gradients, for example). These determinations may be establishedempirically using standard techniques in the art.

For the purpose of aiding in the diagnosis of or predicting amacrophage-associated non-HIV associated dementia, the level ofproliferating macrophages in a sample is generally compared with themean or median level in samples taken from healthy individuals, matchedwhere necessary for sex and age. The level can be calculated as theabsolute number of proliferating macrophages obtained from a bloodsample (or detected by immunohistopathology of a tissue section). Moreusually, the level is calculated as a percentage of total macrophages inthe sample, identifiable by cell markers or morphologicalcharacteristics, since this normalizes for differences in the number ofmacrophage-like cells recovered in the sample.

As with many clinical tests, a finding of about three standarddeviations above the average is statistically significant and indicatesan abnormality. A finding of one or two standard deviations above theaverage is reason for concern. In combination with other indicators, anelevated level may aid in diagnosis of dementia, or some other conditionassociated with macrophage proliferation. For example, peripheral bloodleukocytes stained and counted for PCNA/CD14 cells are consistent withmacrophage-associated dementia in the individual if the percentage ofpositively stained cells is above 50%, more strongly suggestsmacrophage-associated dementia if above about 75%, and even morestrongly suggestive of macrophage-associated dementia if above about90%. The differential diagnosis will include any condition associatedwith macrophage proliferation as a causative or consequential effect,with the ultimate diagnosis being the responsibility of the managingphysician or clinician.

For the purpose of monitoring the effect of a macrophage proliferationinhibitor, the level of proliferating macrophages in a treated sample isgenerally compared with the level in an untreated sample. For thegeneral screening of proliferation inhibitors, peripheral bloodleukocytes are isolated from an individual affected with a diseaseassociated with proliferating macrophages. Samples of the cells aretreated with the candidate compound, and the effect is compared withcells not treated. When administered to a patient, the effect of amacrophage proliferation inhibitor is determined by comparing the levelof proliferating macrophages before and during treatment, with adownward trend generally being consistent with a positive effect.

In another embodiment, the invention provides methods of delayingdevelopment of a macrophage-associated dementia other than AD, in anon-HIV-infected individual (i.e., a non HIV associated dementia otherthan AD). These methods comprise administration of an effective amountof an agent which modulates macrophage proliferation to the individual.Such agents, which include polyamine analogs (including stereoisomers,their salts, and protected derivatives thereof), are described below.The invention also includes methods of treatment or palliation of thesedisorders using an agent(s) which modulates macrophage proliferation.

Agents for Modulating Macrophage Proliferation

In some embodiments of the invention, macrophage proliferation isaccomplished by using a polyamine analog (including stereoisomers,salts, and protected derivatives thereof). In other embodiments, moreparticularly, those which involve a non-HIV associated dementia otherthan AD, any agent which modulates macrophage proliferation may be used.With respect to polyamine analogs, it is understood that the discussionalso applies to stereoisomers, salts and protected derivatives thereof.

Polyamine Analogs

The polyamine analogs used in the present invention include compounds ofthe structures 1, 2, 3, 4, and 5, and the corresponding stereoisomers,salts, and protected derivatives thereof:

-   where R₁, R₂, R₄, R₆ and R₇ are independently selected from the    group consisting of hydrogen, alkyl and aryl, and where R₃ and R₅    are alkyl groups;

-   where R₁, R₂, R₄, R₆, R₈, and R₉ are independently selected from the    group consisting of hydrogen, alkyl and aryl, and where R₃, R₅ and    R₇ are alkyl groups;

-   where R₁, R₂, R₄, R₆, R₈, R₁₀ and R₁₁ are independently selected    from the group consisting of hydrogen, alkyl and aryl, and where R₃,    R₅, R₇ and R₉ are alkyl groups;

-   where R₁ and R₅ are independently selected from the group consisting    of methyl, ethyl, n-propyl, and isopropyl;-   where R₂, R₃, and R₄ are independently selected from the group    consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₃-C₆ cycloalkyl, C₁-C₆    alkyl-C₃-C₆ cycloalkyl-C₁-C₆ alkyl, C₃-C₁₀ aryl, and C₁-C₆    alkyl-C₃-C₁₀ aryl-C₁-C₆ alkyl;-   and where R₆, R₇, R₈ and R₉ are independently selected from the    group consisting of H, methyl, and ethyl;

-   where R₁ and R₆ are independently selected from the group consisting    of methyl, ethyl, n-propyl, and isopropyl;-   where R₂, R₃, R₄ and R₅ are independently selected from the group    consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₃-C₆ cycloalkyl, C₁-C₆    alkyl-C₃-C₆ cycloalkyl-C₁-C₆ alkyl, C₃-C₁₀ aryl, and C₁-C₆    alkyl-C₃-C₁₀ aryl-C₁-C₆ alkyl;    and where R₇, R₈, R₉, R₁₀ and R₁₁ are independently selected from    the group consisting of H, methyl, and ethyl.

Preferably, the polyamine analogs will include compounds of thestructures 2 and 3, where R₃, R₅, R₇ and R₉ are independently (CH₂)_(x)groups, where x is an integer from 2 to 6, and further where R₄, R₆ andR₈ are hydrogen atoms.

More preferably, the polyamine analogs will include compounds of thestructures 2 and 3, where R₃, R₅, R₇ and R₉ are independently (CH₂)_(x)groups, where x is an integer from 2 to 6, and where R₄, R₆ and R₈ arehydrogen atoms, and where R₁ and R₁₀ are alkyl groups, and further whereR₂ and R₁₁ are hydrogen atoms.

Most preferably, the polyamine analogs will include compounds of thestructures 2 and 3, where R₃, R₅, R₇ and R₉ are independently (CH₂)_(x)groups, where x is an integer from 2 to 6, and where R₄, R₆ and R₈ arehydrogen atoms, and where R₁ and R₁₀ are alkyl groups, and where R₂ andR₁₁ are hydrogen atoms, and further where the polyamine analogs have amolecular weight less than 500.

Additional preferred compounds also include compounds of the structure4,

-   where R₆, R₇, R₈ and R₉ are H;-   where R₁ and R₅ are ethyl;-   where R₆, R₇, R₈ and R₉ are H and R₁ and R₅ are ethyl; and/or where    R₂ and R₄ are independently selected from the group consisting of    C₁-C₆ alkyl and R₃ is selected from the group consisting of C₁-C₆    alkyl, C₂-C₆ alkenyl, C₃-C₆ cycloalkyl, C₁-C₆ alkyl-C₃-C₆    cycloalkyl-C₁-C₆ alkyl, C₃-C₁₀ aryl, and C₁-C₆ alkyl-C₃-C₁₀    aryl-C₁-C₆ alkyl.

Additional polyamine analogs useful in the present invention includecompounds of the formula 6, and the corresponding stereoisomers, salts,and protected derivatives thereof:

where R₄ is C₂-C₆ n-alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, orC₃-C₆ aryl;

R₃ and R₅ are independently chosen from a single bond, C₁-C₆ alkyl, orC₁-C₆ alkenyl;

R₂ and R₆ are independently chosen from C₁-C₆ alkyl, C₁-C₆ alkenyl,C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, or C₃-C₆ aryl;

R₁ and R₇ are independently chosen from H, C₁-C₆ alkyl, or C₂-C₆alkenyl; and

R₈, R₉, R₁₀, and R₁₁ are H.

In preferred embodiments of the compounds of formula 6, R₁ and R₇ areindependently chosen from C₁-C₆ alkyl or C₂-C₆ alkenyl.

Additional polyamine analogs useful in the present invention includecompounds of the formula 7, and the corresponding stereoisomers, salts,and protected derivatives thereof:

where R₄ is C₁-C₆ n-alkyl or C₁-C₆ branched alkyl;

R₃ and R₅ are independently chosen from a single bond or C₁-C₆ alkyl;

R₂ and R₆ are independently chosen from C₁-C₆ alkyl, C₁-C₆ alkenyl,C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, or C₃-C₆ aryl;

R₁ and R₇ are independently chosen from H, C₁-C₆ alkyl, or C₂-C₆alkenyl; and

R₈, R₉, R₁₀, and R₁₁ are H.

In preferred embodiments of the compounds of formula 7, R₁ and R₇ areindependently chosen from C₁-C₆ alkyl or C₂-C₆ alkenyl, R₄ is C₁-C₆saturated n-alkyl or C₁-C₆ saturated branched alkyl, and R₃ and R₅ areindependently chosen from a single bond or C₁-C₆ saturated n-alkyl.

When compounds of formulas 1-7 contain terminal primary amino groups(that is, in compounds of formula 1, when R₁ and R₂ are both H, and/orR₆ and R₇ are both H; in compounds of formula 2, when R₁ and R₂ are bothH, and/or R₈ and R₉ are both H; in compounds of formula 3, when R₁ andR₂ are both H, and/or R₁₀ and R₁₁ are both H; in compounds of formula 4,when R₁ and R₆ are both H, and/or R₅ and R₉ are both H; in compounds offormula 5, when R₁ and R₇ are both H, and/or R₆ and R₁₁ are both H; incompounds of formula 6, when R₁ and R₈ are both H, and/or R₇ and R₁₁ areboth H; in compounds of formula 7, when R₁ and R₈ are both H, and/or R₇and R₁₁ are both H), the diseases treated with such compounds includeall diseases disclosed herein except Alzheimer's disease.

Preferably, all the nitrogens of the polyamine analog are independentlysecondary, tertiary, or quarternary amino groups.

Among polyamine analogs preferred for use in this invention are thosecompounds with a demonstrated ability to modulate naturally occurringpolyamine levels in cells. Without intending to be limited by theory,possible mechanisms include competition in the polyamine synthesispathway; upregulation of polyamine catabolizers such as SSAT; affectingpolyamine metabolism.

Of special interest are the following polyamine analogs:

-   1,11-bis(ethyl)norspermine (1,1′-bis(ethylamino)-4,8-diazaundecane;    BE-3-3-3)-   1,8-bis(ethyl)spermidine (BES)-   1,12-bis(ethyl)spermine (BESm; DESPM (N¹,N¹²-diethylspermine;    SunPharm);-   1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4)    (Diethylhomospermine, N¹,N¹⁴-diethylhomospermine; DEHOP or DEHSPM;    SunPharm)-   diethyl-norspermine (DENOP; SunPharm)-   1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4)-   N-ethyl-N′-(2-(3′-ethylamino-propylamino    methyl)-cis-cyclopropylmethyl)-propane 1,3-diamine    tetrahydrochloride (SL-11037), provided by S'LIL, Madison, Wis.-   N-ethyl-N′-(2-(3′-ethylamino-propylamino    methyl)-trans-cyclobutylmethyl)-propane 1,3-diamine    tetrahydrochloride (SL-11038), S'LIL-   N-ethyl-N′-(2-(3′-ethylamino-propylamino    methyl)-trans-cyclopropylmethyl)-propane 1,3-diamine    tetrahydrochloride (SL-11044), S'LIL.-   N,N′-bis(3-ethylaminopropyl)-cis-but-2-ene-1,4-diamine    tetrahydrochloride (SL-11047), S'LIL    The structures of SL-11037, SL-11038, SL-11044, and SL-11047 are    diagrammed below:

Besides the polyamine analogs listed above, stereoisomers, salts orprotected derivatives thereof, may be used. The invention also comprisesmethods of using an effective amount of any of the polyamine analogslisted above, or stereoisomers, salts or protected derivatives thereof(or a composition comprising an effective amount of any of the polyamineanalogs listed above, or stereoisomers, salts or protected derivativesthereof) in modulating macrophage proliferation (or in treating ordelaying development of macrophage-associated diseases, includingHIV-associated dementia and AD). The invention also comprises anypolyamine analog listed above, or stereoisomers, salts or protectedderivatives thereof, for use in preparing compositions (i.e.,medicaments) useful for treating macrophage-associated diseases,including HIV-associated dementia, and Alzheimer's disease.

Any polyamine analog listed above, or stereoisomers, salts or protectedderivatives thereof (or a composition comprising an effective amount ofany polyamine analog listed above, or stereoisomers, salts or protectedderivatives thereof) may be used in vitro or in vivo. In vitro, asuitable biological sample (such as a blood sample, which may or may notbe enriched for the macrophage population) is contacted with thecomposition(s). In vivo, a composition of the invention is generallyadministered according to the manufacturer's/supplier's instructions.Generally, polyamine analogs are administered by subcutaneous orintravenous injection. They may also be administered orally.

The amount of a polyamine analog (or stereoisomers, salts or protectedderivatives thereof) administered will depend on several variables, suchas the particular analog (or sterioisomer, salt or protectivederivative) used, the time course of administration, the condition ofthe individual, the desired objective, the extent of disease, how manydoses will be administered, and whether any other substances are beingadministered. Generally, the amount used will be as recommended by themanufacturer and/or based on empirical studies. In the case of polyamineanalogs (or stereoisomer, salt, or protected derivative thereof), theamount will generally be between about 1 to about 300 mg/m²/day,possibly between about 15 to about 150 mg/m²/day. Administration isgenerally intermittant, meaning that analog (or stereoisomer, salt, orprotected derivative thereof) is administered per a period of at leastone to two days and then not administered for a period of at least oneto two days, with the cycle repeated as indicated. In one embodiment,the polyamine analog (or stereoisomer, salt, or derivative thereof) for6 days every three weeks.

Routes of administration will generally depend on the nature of theparticular polyamine analog (or stereoisomer, salt or protectivederivative) used, and may be, for example, oral or by injection(subcutaneous or intravenous). Administration is generally byintravenous or subcutaneous injection.

Preferably, a polyamine analog (or stereoisomer, salt or protectedderivative), or other suitable agent that interferes with the polyaminesynthetic pathway, polyamine metabolism, and/or the intracellularconcentration maintenance of spermine) is administered in a suitablepharmaceutical excipient. Pharmaceutical excipients are known in the artand are set forth in Remington's′ Pharmaceutical Sciences, 18th edition,Mack Publishing (1990). The polyamine analog may also be associated withanother substance that facilitates agent delivery to macrophages, orincreases specificity of the agent to macrophages. For example, anagent(s) may be associated into liposomes. Liposomes are known in theart. The liposomes in turn may be conjugated with targetingsubstance(s), such as IgGFc receptors. Substances that increasemacrophage phagocytosis such as zymosan or tetrachlorodecaoxygen (TCDO)and/or activation such as MCSF, GMCSF or IL-3 may be used to increaseuptake of anti-proliferative agent(s).

A polyamine analog (or stereoisomer, salt or protected derivative) maybe administered alone, or in conjunction with other substances and/ortherapies, depending on the context of administration (i.e., desired endresult, condition of the individual, and indications). “In conjunctionwith” means that an agent is administered prior to, concurrently, orafter other substance or therapy. Examples of substances that might beadministered in conjunction with an agent include, but are not limitedto, brain neurochemical modulators (in the context ofmacrophage-associated dementias), and classic anti-neoplastic agentsand/or anti-lymphocytic agents such as steroids and cyclosporinederivatives. For example, a polyamine analog (or a stereoisomer, salt orprotected derivative thereof) can be administered in conjunction withmitoguazone dihydrochloride.

The mechanistic effectiveness of various polyamine analogs and enzymeinhibitors can be determined at least in part by their ability todeplete intracellular polyamine pools. Kramer et al. [(1995) Biochem.Pharmacol. 50:1433] describe the use of 4-fluoro-L-ornithine to monitormetabolic flux through the polyamine biosynthetic pathway. It wasdetermined that the metabolic flux indicated by the rate of appearanceof fluorinated polyamines, reflected the proliferation status of thecells. U.S. Pat. No. 5,498,522 outlines the use of SSAT as a prognosticindicator or tumor response marker. Either SSAT enzyme activity, SSATenzyme protein, or mRNA transcripts can be measured directly, or otherdeterminants related to SSAT induction can be measured, such as SSATco-factor acetylCoA, and the SSAT products N1-acetylspermine andN1-acetylspermidine. To further determine the effect of a polyamineanalog's administration, an individual may be monitored for disease (orprecursor disease) progression as well as biochemical and/or geneticmarkers of disease (or precursor disease). With respect to diseaseprogression, multiple rating scales (i.e., indices of clinical function)have been established and are known in the art for various macrophageproliferative disorders such as AD and lymphomas. Formacrophage-associated neurological disorders, cognitive functions can betested and, in some cases, imaging modalities such as MRI may be used.

Other Agents for Modulating Macrophage Proliferation

Besides the polyamine analogs described above, suitable agents for usein modulating macrophages in the context of non-HIV associated dementiasother than AD, include general anti-proliferative agents (i.e.,proliferation-modulating agents). These include, but are not limited to,daunomycin, mitomycin C, daunrorubicin, doxorubicin, 5-FU, cytocinearabinoside, colchicine, cytochalasin B, bleomycin, vincristin,vinblastine, methotrexate, cis platinum, ricin, abrin, diphtheria toxin,and saporin.

Other suitable agents would be those which inhibit, or interfere with,the polyamine synthetic pathway, or those which affect the metabolism ofpolyamines. Other suitable agents are those which affect the closelyregulated intracellular concentration of spermidine. An example of suchan agent is MGBG (mitoguazone dihydrochloride; XYRKAMINE®; Ilex, Texas)which inhibits S-adenosylmethionine decarboxylase which in turn isrequired for the production of polyamines. Any agent that interfereswith polyamine interactions with proliferating macrophage target, suchas DNA, RNA, and/or membranes would likewise be suitable. Another typeof useful agent is one that interferes with polyamine interactions withDNA. Such an agent(s) could exert this function, for example, by any ofthe effects above (i.e., interfering with the polyamine syntheticpathway and/or metabolism, disturbing the concentration of intracellularspermine, competitors, etc.) as well as affecting polyamine function interms of interacting with DNA. It is understood that, with respect tothese and any other agent described herein, toxicology considerationsalso must be taken into account when determining whether, and/or in whatamount, an agent is to be used.

It is understood that, with respect to the above-described agents, somecan reasonably be considered as, and are considered as, polyamineanalogs. An example is MGBG.

Administration and other considerations have been described above.

The following examples are provided to illustrate but not limit theinvention.

EXAMPLES Example 1 Testing Various Polyamine Analogs for Effect onMacrophage Proliferation in the Context of Macrophage-AssociatedDisorders Separation of Peripheral Blood Monocytes

We had previously separated peripheral blood monocytes on Ficoll. Wefound that a standard Ficoll Hypaque separation gave lower yields ofmonocytes from demented patients than from HIV-infected controls (44 vs67%, respectively). Several demented patients yielded under 10% ofpredicted, suggesting that a subset of monocytes was being missed. Wethen used Percoll gradient separation, which allows for denser cells tobe captured. A two-step gradient was prepared in 15 mL conical tubes:bottom layer of 1.087 density Percoll, overlaid with 1.077 density. 1.5mL whole heparinised blood was mixed with an equal volume of isotonicsaline. This blood/saline was layered over the gradient and centrifuged.Cells from the 1.077 and 1.087 interfaces were collected, combined, andwashed in 5 volumes of RPMI 1640. Specimens from patients withmacrophage proliferative diseases such as AIDS dementia had a highfrequency of dense (1.087 g/cc) monocytes. All further experiments wereperformed using 1.087 gradient.

Five×10⁵ PBMC's isolated through a 1.087 g/cc Percoll/saline gradientwere exposed to varied concentrations of agent after baseline CD14/PCNAstaining was performed. The cells were cultured at 5×10⁵ cells inRPM1-1640/10% fetal calf serum in a polypropylene tube (Falcon) at 37°C. for five days. After five days CD14/PCNA staining was performed oncontrol and agent-treated cultures. FACSCAN analysis of PCNA-positivecells in control cultures was compared with agent-treated cultures andthe percentage of control PCNA/CD14 cells was calculated. The results ofsuch an experiment in which the effect of polyamine analog DEHOP(SunPharm; Florida) on proliferating macrophages (as detected by PCNA)from the blood of four patients with AIDS dementia are shown in FIG. 1A(with underlying data provided in Table 3). This polyamine analogsignificantly reduced the percentage of proliferating macrophages. Acomparison of the inhibitory effect of DEHOP and cyclosphosphamide,another anti-proliferative agent, was performed in vitro using the bloodof one of these patients (“CB”). The results are shown in Table 1. Thisexperiment showed that DEHOP was superior in inhibition. This polyamineanalog effect is relative and specific as indicated by a lack of invitro toxicity in this assay system by cyclosphosphamide, a classicanti-neoplastic agent.

A comparison of polyamine analogs SL-11037, SL-11038 (S'LILPharmaceuticals) and DENOP (SunPharm) on macrophage proliferation fromthe blood of one patient with AIDS dementia is shown in Table 2. All 3agents showed inhibitory effects. FIG. 1B similarly shows that polyamineanalog BE-4444 demonstrates potent killing of PCNA⁺/CD14⁺ cells. Thedata shown in FIG. 5 similarly demonstrate that polyamine analogsSL-11047, SL-11048 and SL-11044 (S'LIL Pharmaceuticals) are potentinhibitors of PCNA⁺ macrophage detection from peripheral blood of HIV⁺sarcoid patient. 60% of input CD14 cells were PCNA⁺; the assay wasperformed 96 hr after drug exposure and culture. The data shown in FIG.6 demonstrate that polyamine analogs DEHOP and SL-11047 all decreasePCNA⁺/CD14 cell survival. The data presented are averages of several (N)experiments performed. Thus, the data shown herein demonstrate thatBE-4444, SL-11037, SL-11038, SL-11044, SL-11047, SL-11048, DEHOP andDENOP all demonstrated potent inhibition and/or killing of bloodPCNA⁺/CD14⁺ cells.

TABLE 1 Effect of DEHOP and Cyclophosphamide (Cytoxan ®, Bristol-MyersSquibb) on CD14⁺, PCNA⁺ peripheral blood macrophage from an HIV dementiapatient #cells/10,000 PBMC CD14⁺/PCNA⁺ CD14⁺/PCNA⁻ total CD14 DEHOPConcentration DEHOP 0 74 472 546 10 um 31  89 120 2 28 182 210 0.4 25222 247 Cyclophosphamide Concentration Cyclophosphamide 0 74 472 546 5μg/ml 70 272 342 0.5 49 390 439 0.05 99 455 554 0.005 71 295 366 PatientCB at time 0: % of CD14⁺ that were CD69⁺: 0 % of CD14⁺ that were PCNA⁺:28% median SSC for all CD14⁺: 420

TABLE 2 EFFECTS OF DRUGS SL-11037, SL-11038 AND DENOP ON CD14⁺, CD69⁺AND CD14⁺, PCNA⁺ CELLS * NO. OF CELLS/5,000 SL-11038 DENOP SL-11037 5010 0 50 10 2

M 10 2 50

M 2 DRUG

M

M

M *

M

M

M *

M CD14⁺, CD69⁺ 35/5000 33/5000 49/5000 46/5000 26/5000 32/5000  30/500032/5000 23/5000 32/5000 CD14⁺, PCNA⁺ 64/5000 11/5000 32/5000 49/500024/5000 36/5000 104/5000 19/5000 23/5000 34/5000 TOTAL 388 79 116 276104 205 356 43 50 149 CD14/PCNA

TABLE 3 In-vitro inhibition of CD14⁺/PCNA antigen expression onperipheral a blood macrophage from HIV dementia patients % InhibitionConcentration DEHOP (um) patient 50 10 2 0.4 0.08 1 84 95 79 2 97 94 943 58 62 66 4 75 25 19 mean 84 83 +/− 22 78 +/− 13 62 +/− 34 19

Example 2 Suppression of In Vitro Cell Killing by Supernatants fromPeripheral Blood from an AIDS Dementia Patient

We have previously observed a cell killing effect of mononuclear cellsupernatants from peripheral blood of patients with AIDS dementia.Pulliam et al. (1997) Lancet 349: 692-695. We tested a similarlyprepared supernatant which was treated with the polyamine analog DEHOP.

Preparation of Mononuclear Cell Supernatants

To collect a maximum number of monocytes (both heavy and light), we useda one-step Percoll protocol. 1.5 mL whole heparinised blood was mixedwith an equal volume of isotonic phosphate-buffered solution and layeredover 5 mL Percoll at 1.087. The cells were centrifuged, washed, andresuspended in RPMI 1640 supplemented with 10% fetal calf serum at10°/mL. To separate non-adherent lymphocytes from adherent monocytes,culture dishes were washed with RPMI after overnight incubation. Theadherent cells were re-fed with RPMI and cultured for 7 days to obtainsupernatants. Supernatants were clarified by centrifugation at 90,000 gover 25% sucrose overnight. The supernatant was filtered through a 0.22

m syringe filter and stored at −70° C.

Brain Aggregate Cultures and Supernatant Treatment

Human fetal brain tissue from between 16 and 18 weeks' gestation wasobtained from elective abortions for preparation of brain cellaggregates. These aggregates contain all the cells of the centralnervous system including neurons, astrocytes, oligodendrocytes withaccompanying myelin, and microglial cells. After 10 days in culture, andbefore experimentation, aggregates were tested for viability bytrypan-blue exclusion. Aggregates were divided into flasks at 50 mgwet-weight in 2 mL exchange medium (DMEM with 15% fetal calf serum).Aggregates were treated with mononuclear cell supernatants (20%) for 3days. The flasks contents were centrifuged; the supernatant was used forlactate dehydrogenase assay and the aggregate pellet for DNAfragmentation (programmed cell death) assay.

Quantification of Cell Death by ELISA

Lactate dehydrogenase is released on cell lysis. We used a cytotoxicityELISA kit (Boehringer Mannheim). The brain aggregate supernatant wasreacted with yellow tetrazolium salt and absorbance was read at 490 nm.The DNA fragmentation assay kit (Boehringer Mannheim) measures cytosolicoligonucleosome-bound DNA. The brain aggregate pellet was solubilisedand used for this assay.

Electron Microscopy

Brain cell aggregates were treated with 20% supernatant from culturedCD14 cells from a demented AIDS patient. Treatment was for 72 h. Aftertreatment, the aggregates were washed and fixed in Karnovsky's solution.The cells were post-fixed in 1% osmium tetroxide dehydrated for stainingwith uranyl acetate and lead citrate. Thin sections were examined in aJEOL 100SX electron microscope.

The results are shown in FIG. 2. Untreated macrophage supernatants fromperipheral blood an AIDS dementia patient showed significant cellkilling, which virtually disappeared if the preparations were treatedwith DEHOP. Non-dementia disease control did not show significant cellkilling, either with or without DEHOP treatment.

Example 3 Detection of Proliferating Macrophages in Patients withAlzheimer's Disease Participants and Methods Blood Samples

Individuals with Alzheimer's disease dementia (6 patients) wererecruited from Laguna Honda hospital, University of California at SanFrancisco, and San Francisco General hospital. Exclusion criteriaincluded history of head injury, seizures, or multiple sclerosis, activeopportunistic infection, active opportunistic central nervous systeminfection or lymphoma, cerebrovascular disease, major psychiatricillness, other known causes of dementia, or pre-existing causes of braindisorder. Participants or their representatives gave informed consent.Blood was obtained in accordance with the Committee on Human Research.Negative controls (6 patients) were recruited from laboratory personneland gave informed consent. Non-AD diseased control categories weremultiple sclerosis (2 patients), amyotrophic lateral sclerosis (2patients), AIDS dementia (7 patients), and Parkinson's disease (2patients).

Flow Cytometry for Monocyte Subsets

5 mL blood was obtained from patients and controls. 100 μL wholeheparinised blood was stained with fluorescein-conjugated anti-CD14(DAKO Corp.) and with phycoerythrin-conjugated anti-PCNA (BectonDickinson). Red-blood cells were lysed by the addition of FACSLYSEsolution (Becton Dickinson). The cell suspensions were centrifuged andthe cell pellets resuspended in phosphate-buffered saline containingsodium azide and paraformaldehyde and stored at 4° C. To stainintracellularly for PCNA, cell pellets were resuspended in“Permeabilising Solution” (Becton Dickinson). Buffer was added to eachtube, followed by centrifugation. Cell pellets were resuspended inbuffer and stained with phycoerythrin-conjugated PCNA. The cells werewashed, fixed, and stored as above.

Cells were analysed with a FACSCAN flow cytometer (Becton Dickinson). Atleast 2000 cells per sample were analysed. Negative staining was definedas the area of dot plots that contained over 99% of isotype-stained(DAKO) “control cells”. The results are shown in FIG. 4. The patientswith AD displayed a significantly higher percentage of proliferatingmacrophages or non-disease controls as well as diseased controls,including AIDS dementia. Individual values (percentage PCNA/CD14 cells)were: (a) non-disease controls, 10; 16; 19; 20; 27; 46; (b) diseasecontrols: ALS, 17; 25; Parkinson's, 27; 45; MS, 7; 20; HIV dementia, 6;28; 37; 52; 54; 61; 96; (c) AD patients, 44; 47; 91; 97; 98; 100.

Thus, the data presented herein show that macrophage proliferation ischaracteristic of Alzheimer's disease.

Example 4 Effect of Polyamine Analogs on PCNA Expression of PeripheralMacrophages from Patients with AD Separation of Peripheral BloodMonocytes

We had previously separated peripheral blood monocytes on Ficoll. Wefound that a standard Ficoll Hypaque separation gave lower yields ofmonocytes from demented patients than from HIV-infected controls (44 vs67%, respectively). Several demented patients yielded under 10% ofpredicted, suggesting that a subset of monocytes was being missed. Wethen used Percoll gradient separation, which allows for denser cells tobe captured. A two-step gradient was prepared in 15 mL conical tubes:bottom layer of 1.087 density Percoll, overlaid with 1.077 density. 1.5mL whole heparinised blood was mixed with an equal volume of isotonicsaline. This blood/saline was layered over the gradient and centrifuged.Cells from the 1.077 and 1.087 interfaces were collected combined, andwashed in 5 volumes of RPMI 1640.

Five×10⁵ PBMC's isolated through a 1.087 g/cc Percoll/saline gradientwere exposed to varied concentrations of agent after baseline CD14/PCNAstaining was performed as described in Example 3. The cells werecultured at 5×10⁵ cells in RPM1-1640/10% fetal calf serum in apolypropylene tube (Falcon) at 37° C. for five days. After five daysCD14/PCNA staining was performed on control and agent-treated cultures.FACSCAN analysis of PCNA-positive cells in control cultures was comparedwith agent-treated cultures and percentage of control PCNA/CD14 cellswas calculated. Two polyamine analogs, DENOP (SunPharm; Florida) andSL-11037 were tested. As shown in FIG. 3, both agents significantlyreduced the percentage of proliferating macrophages.

Example 5 Using Polyamine Analog to Treat an Individual with aMacrophage-Associated Dementia

A 72-year-old male presents with increasing loss of memory andconfusion. A blood sample is obtained, and the percentage ofPCNA-expressing CD14 cells is significantly elevated over the control.The patient is given DESPM (a polyamine analog) at 125 mg/m²/day for sixdays, repeated every three weeks. Blood samples are obtained routinelyto monitor the level of PCNA-expressing CD14 cells.

Example 6 Effects of Polyamine Analog diethylhomospermine (DEHOP) onChemotherapy Resistant AIDS Related Lymphoma Patients

Four patients who had failed standard chemotherapy for treatment oftheir AIDS related lymphoma (therapy included at least CHOP chemotherapyup to six cycles) were evaluated for response to DEHOP administration invivo. Patients had blood drawn pre administration of drug for evaluationof macrophage activation. This blood was also evaluated for response toDEHOP in vitro. The trial was set up to evaluate clinical response todrug given for 5 days subcutaneously (sq) at a dose of 25 mg/m² (2patients) and 50 mg/m² (2 patients). Blood was obtained at 1 week aftercompletion of the first and second cycles of therapy. The bloodspecimens were evaluated for macrophage proliferation (proliferatingcell nuclear antigen, PCNA expression), and activation (percentage CD69expression). Response to therapy was evaluated by direct observation oftumor (3 patients) and persistence or resolution of symptoms (1 patientwith GI lymphoma).

To test whether patient macrophages would respond to administration ofDEHOP in vitro, CD14 cells in a peripheral blood mononuclear cellpreparation (PBMC) were exposed to different concentrations of DEHOP andthe proportion of CD14 positive cells surviving at the end of 5 days ofculture was assessed in drug treated cells as compared to controls.Specimens obtained pre chemotherapy were compared to specimens obtained2 weeks post therapy. Table 4 summarizes CD14⁺ cell responses to DEHOPin vitro in the 4 AIDS lymphoma patients evaluated; FIG. 7 shows theCD14⁺ cell response to DEHOP in vivo. For patient FL, the first specimenwas obtained one day after the first cycle of DEHOP; the second specimenwas obtained one week later. The effective dose 50 (where 50% of cellswere killed), and effective dose 80 (where 80% of cells were killed)were calculated for each set of patient specimens exposed to DEHOP invitro. In post in vivo DEHOP administration blood specimens, patient FTJwas persistently resistant to drug whereas patient LT and FL, both ofwhom responded clinically to DEHOP, showed marked drug sensitivity invitro.

To determine whether kinetics of macrophage activation parameterspredicted response to DEHOP therapy, CD14 cell analysis results from the2 patients who had demonstrated a clinical response were combined andplotted in FIG. 7. FIG. 7 shows that both patients showed elevatedlevels of PCNA/CD69 expression at initiation of therapy, a decreaseduring tumor response and an increase just prior to tumor relapse. Thesevalues generally decreased after administration of drug (shown in thedark shaded box along the x-axis) and a low to background level ofexpression of all parameters was observed for both patients on day 35after initiation of study. Both patients had objective and subjectiveresponses to DEHOP at this time. Because of intercurrent medicalproblems the patients were not allowed to receive a third cycle of DEHOPand both patients ultimately relapsed, FL on day 47 and LT on day 54.All of the macrophage activation parameters increased in both patientsprior to their clinical objective tumor relapse.

In contrast to the 2 patients who responded to DEHOP clinically, wheremacrophage activation parameters seemed to predict sensitivity andresponse to drug, patient FTJ and WA showed no change in PCNA expressionor CD69 expression after the first cycle of DEHOP and had no tumorresponse to drug.

The information obtained by evaluating drug sensitivity of patientmacrophages in vitro to DEHOP exposure and tumor response to DEHOP invivo suggests that response of the macrophages to DEHOP eithercorrelated or showed some association with tumor sensitivity to DEHOPtreatment in vivo. These data suggest that sensitivity to the polyamineanalog DEHOP predicts tumor response to the drug and that at time ofrelapse the re-expression of activated/proliferating macrophagescorrelates with that tumor response. One interpretation of this data isthat if patients have circulating macrophages that areactivated/proliferating that respond to DEHOP, those patients may becandidates for a response to the drug in vivo.

TABLE 4 CD14 Cell Polyamine Analog Sensitivity: In vitro dose responsein NHL patient PBMC; total CD14⁺ cell survival, ED₅₀ and ED₈₀ PatientSpecimen ED_(50 μm) ED_(80 μm) Response to Therapy FTJ Pre 1.0 >10 NonePost >10 >10 WWA Pre 10 >10 None LT Pre 10 >10 Clinical Response Post0.1 0.8 FL Pre <0.1 1.0 >50% objective measured tumor Post <0.1 0.1response after first week of Rx

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity of understandingit will be apparent to those skilled in the art that certain changes andmodifications will be practiced. Therefore, the description and examplesshould not be construed as limiting the scope of the invention, which isdelineated by the appended claims.

1.-44. (canceled)
 45. A method of treating a subject afflicted with, orat risk for, atherosclerosis or glomerulosclerosis, or delayingdevelopment or onset of atherosclerosis or glomerulosclerosis in saidsubject comprising administering to said subject effective amount of acomposition comprising mitoguazone or a salt of mitoguazone.
 46. Themethod of claim 45, wherein the composition comprises mitoguazonedihydrochloride.
 47. A method of modulating macrophage proliferation inan individual afflicted with, or at risk for, a macrophage-associateddisorder comprising administering to the individual an effective amountof a composition comprising mitoguazone or a salt of mitoguazone,wherein the disorder is atherosclerosis or glomerulosclerosis.
 48. Themethod of claim 47, wherein the composition comprises mitoguazonedihydrochloride.
 49. A method of treating a subject afflicted with, orat risk for, a macrophage-associated disorder or delaying onset ordevelopment of a macrophage-associated disorder in said subjectcomprising administering to said subject an effective amount of acomposition comprising mitoguazone or a salt of mitoguazone to reducemacrophage proliferation in said subject, wherein the disorder isatherosclerosis or glomerulosclerosis.
 50. The method of claim 49,wherein the composition comprises mitoguazone dihydrochloride.